This invention relates to an anti-Fas ligand antibody and an assay method utilizing the anti-Fas ligand antibody. More specifically, this invention relates to a method for assaying a Fas ligand in human body fluid utilizing an anti-Fas ligand antibody, and the antibody adapted for use in such an assay. This invention also relates to an anti-Fas ligand antibody that exhibits high suppression of the Fas ligand-induced apoptosis. This invention also relates to a hybridoma or a cell line that produces such an antibody.
Human Fas ligand is a polypeptide that has been reported by Nagata et al. to be a biological molecule that induces apoptosis of Fas antigen-expressing cells (Takahashi, T. et al., International Immunology, vol. 6, 1567-1574, 1994). Human Fas ligand is a Type II membrane protein of TNF family with a molecular weight of about 50 kD. As in the case of TNF, human Fas ligand in the human body is estimated to be in the form of a trimer (Tanaka, M. et al., EMBO Journal, vol. 14, 1129-1135, 1995). The extracellular domain of the human Fas ligand is highly homologous with the extracellular domain of rat Fas ligand (Suda, T. et al., Cell, vol. 75, 1169-1178, 1993) and mouse Fas ligand (Takahashi, T. et al., Cell, vol. 76, 969-976, 1994). The human Fas ligand recognizes not only human Fas antigen but also mouse Fas antigen, and the apoptosis is induced after such recognition of the Fas antigen. At the same time, the rat Fas ligand and the mouse Fas ligand recognize the human Fas antigen to induce the apoptosis.
Apoptosis has called attention for its deep involvement in homeostasis of an organism. The homology of the Fas ligands among different species as mentioned above suggests the important role of the apoptosis mediated by the Fas ligand and the Fas antigen in the homeostasis of organism.
Recently, an interesting relation of abnormality of Fas ligand and Fas antigen with an autoimmune disease has been reported. In this report, there is suggested that MRL-lpr/lpr (a strain of model mouse for an autoimmune disease) has mutation in its Fas antigen gene, and apoptosis is not induced in the cells expressing such mutant Fas antigen gene (Watanabe-Fukunaga, R. et al, Nature, vol. 356, 314-317, 1993; Adachi, M. et al., Proc. Natl. Acad. Sci. USA, vol. 90, 1993). In the meanwhile, there has also been reported that C3H-gld/gld (another strain of model mouse for an autoimmune disease) has mutation in its Fas ligand gene, and that the Fas ligand of the gld mouse has no apoptosis-inducing activity. The mutation in the Fas ligand gene of the gld mouse is a point mutation, and as a result of such point mutation, 7th amino acid from the C terminal of the extracellular domain of the Fas ligand is replaced with another amid acid (Takahashi, T. et al., Cell, vol. 76, 969-976, 1994). The Fas ligand the gld mouse as described above is incapable of binding with the Fas antigen (Ramsdell, F. et al., Eur. J. Immunol., vol. 24, 928-933, 1994).
The findings as described above resulted in the hypothesis that some autoimmune diseases are induced by the abnormality of the Fas antigen or the Fas ligand, namely, by the autoreactive T cells remaining in the body that should have been removed from the body by undergoing apoptosis if the cells had been normal.
Recently, it is conceived that abnormal propagation of synovial membrane that takes place in rheumatism is also induced by the failure of normal apoptosis of the cell. Kobayashi, N. et al. further estimates that reduction in the number of T cells upon infection by AIDS virus is mediated by the Fas ligand since expression of the Fas antigen on the T cell membrane is induced upon infection by the AIDS virus (Nikkei Science, vol. 6, 34-41, 1993).
As the relation between the apoptosis mediated by the Fas ligand-Fas antigen and the diseases is gradually found out, a growing expectation is present for the use of the Fas ligand or the Fas antigen in the treatment of the diseases associated with abnormality of the apoptosis, such as the above-mentioned autoimmune diseases, rheumatism and AIDS.
Nagata et al. have also reported that they succeeded in obtaining an antibody against the Fas ligand, and that such antibody was capable of suppressing the apoptosis (Tanaka, M. et al., EMBO Journal, vol. 14, 1129-1135, 1995; International Patent Application Laid-Open No. WO95/13293).
Nagata et al. have made further investigation on an N terminal-deleted Fas ligand and succeeded in creating a Fas ligand with higher activity (International Patent Application No. PCT/JP95/00883).
As described above, Nagata et al. have made a series of intensive studies to isolate the Fas ligand, and prepared an antibody against such Fas ligand.
However, there has so far been no report of actual measurement of the Fas ligand in the human body fluid, and presence of a soluble Fas ligand in the human body fluid has remained unconfirmed. It is generally conceived that a physiologically active cytokine such as Fas ligand is topically produced in a minute amount to topically madiate an action. Such cytokine usually has a short half life, and after mediating an action, the cytokine is rapidly discharged from the tissue or blood. Therefore, a cytokine such as Fas ligand is not readily detectable in peripheral blood. Fas ligand was originally reported as a transmembrane (membrane-bound) protein, and release of the Fas ligand from the cell under some special conditions is indicated in an in vitro experiment (Tanaka, M. et al., EMBO Journal, vol. 14, 1129-1135, 1995). Occurrence of such phenomenon under in vivo, physiological conditions has been unknown, and presence of the Fas ligand in human body fluid has also been unknown. Even if the Fas ligand was present in the human body fluid, the concentration of the Fas ligand has been estimated to be very low, and measurement of the Fas ligand at such a low concentration has been regarded quite difficult. In addition, various immunoassay-interfering substances are likely to be present in a body fluid, and therefore, development of a Fas ligand assay system with a high sensitivity and a high specificity and an antibody adapted for such an assay has been required. In view of the relation between the Fas ligand and various diseases indicated, measurement of the Fas ligand concentration in the body fluid is deemed clinically valuable as a means for diagnosing such diseases. However, there has so far been no report directed to the fluctuation of the Fas ligand concentration in the body fluid. In view of the future administration of the Fas ligand or a substance that may affect the activity or expression of the Fas ligand to patients suffering from a disease for which administration of an apoptosis-inducing substance such as Fas ligand might be effective, it is necessary to enable measurement of the Fas ligand concentration in the body fluid to thereby monitor the blood concentration and determine the therapeutic effects. A Fas ligand assay method of high sensitivity and specificity has thus been awaited. With regard to the neutralizing antibody, a neutralizing antibody of a higher neutralizing activity and clarification of the action mechanism has been awaited, and in view of the therapeutic application, an antibody capable of suppressing the apoptosis at a low dosage has been awaited for both effectivity and safety.
Unfortunately, the use of non-human monoclonal antibodies such as mouse monoclonal antibodies have certain drawbacks in human treatment, particularly in repeated therapeutic regimens as explained below. Mouse monoclonal antibodies, for example, have a relatively short circulating half-life, and lack other important immunoglobulin functional characteristics when used in humans.
Perhaps more importantly, non-human monoclonal antibodies contain substantial stretches of amino acid sequences that will be immunogenic when injected into a human patient. Numerous studies have shown that, after injection of a foreign antibody, the immune response elicited by a patient against an antibody can be quite strong, essentially eliminating the antibody's therapeutic utility after an initial treatment. Moreover, as increasing numbers of different mouse or other antigenic (to humans) monoclonal antibodies can be expected to be developed to treat various disease, after one or several treatments with any different non-human antibodies, subsequent treatments even for unrelated therapies can be ineffective or even dangerous in themselves, because of cross-reactivity.
While the production of so-called "chimeric antibodies" (e.g., mouse variable regions joined to human constant regions) has proven somewhat successful, a significant immunogenicity problem remains. In general, the production of human immunoglobulins reactive with Fas ligand, as with many antigens, would be extremely difficult using typical human monoclonal antibody production techniques. Similarly, utilizing recombinant DNA technology to produce so-called "humanized" or "reshaped" antibodies (see, e.g., Riechmann et al., Nature 332, 323 (1988) and EPO Publication No. 0239400, which are incorporated herein by reference), provides uncertain results, in part due to unpredictable binding affinities of the resultant immunoglobulins. In addition, an antibody that reacts with a particular peptide derived from the Fas ligand, namely, an antibody whose binding site within the Fas ligand has been found out, has been awaited for a further investigation of the Fas ligand and analysis of the Fas ligand function.